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1.
J Biol Chem ; 294(4): 1257-1266, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30514758

RESUMO

Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Técnicas Biossensoriais , Pontos de Checagem do Ciclo Celular/genética , Resistência a Múltiplos Medicamentos/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Genoma Fúngico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
2.
Proc Natl Acad Sci U S A ; 112(45): 13952-7, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26499248

RESUMO

Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.


Assuntos
Evolução Química , Evolução Molecular , Família Multigênica , Policetídeo Sintases/genética , Policetídeos/química , Filogenia
3.
Angew Chem Int Ed Engl ; 56(32): 9556-9560, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28679030

RESUMO

Fungal polyketides have significant biological activities, yet the biosynthesis by highly reducing polyketide synthases (HRPKSs) remains enigmatic. An uncharacterized group of HRPKSs was found to contain a C-terminal domain with significant homology to carnitine O-acyltransferase (cAT). Characterization of one such HRPKS (Tv6-931) from Trichoderma virens showed that the cAT domain is capable of esterifying the polyketide product with polyalcohol nucleophiles. This process is readily reversible, as confirmed through the holo ACP-dependent transesterification of the released product. The methyltransferase (MT) domain of Tv6-931 can perform two consecutive α-methylation steps on the last ß-keto intermediate to yield an α,α-gem-dimethyl product, a new programing feature among HRPKSs. Recapturing of the released product by cAT domain is suggested to facilitate complete gem-dimethylation by the MT.


Assuntos
Carnitina Aciltransferases/metabolismo , Policetídeo Sintases/metabolismo , Trichoderma/enzimologia , Biocatálise , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Domínio Catalítico , Metabolômica , Estrutura Molecular
4.
Fungal Genet Biol ; 89: 18-28, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26808821

RESUMO

Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, "activity-guided" approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent "genome mining" approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias toward the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and synthetic biology efforts toward discovering novel fungal enzymes and metabolites.


Assuntos
Produtos Biológicos , Vias Biossintéticas/genética , Genes Fúngicos , Genoma Fúngico , Família Multigênica , Alcaloides , Sequência de Aminoácidos , Biologia Computacional , Curadoria de Dados , Fungos/genética , Filogenia , Policetídeos , Terpenos
5.
J Am Chem Soc ; 137(43): 13724-7, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26469304

RESUMO

The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.


Assuntos
Diterpenos/metabolismo , Engenharia Genética , Indóis/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Saccharomyces cerevisiae/enzimologia , Vias Biossintéticas/genética , Ciclização , Diterpenos/química , Indóis/química , Conformação Molecular , Saccharomyces cerevisiae/metabolismo
6.
Sci Adv ; 4(4): eaar5459, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29651464

RESUMO

For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.


Assuntos
Produtos Biológicos/metabolismo , Fungos/genética , Fungos/metabolismo , Expressão Gênica , Engenharia Genética , Vias Biossintéticas , Fermentação , Engenharia Genética/métodos , Ensaios de Triagem em Larga Escala , Regiões Promotoras Genéticas , Fluxo de Trabalho
7.
Genetics ; 174(1): 511-8, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16816425

RESUMO

The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast.


Assuntos
Proteínas Fúngicas/metabolismo , Íntrons/fisiologia , RNA Fúngico/metabolismo , Leveduras/genética , Actinas/genética , Deleção de Genes , Duplicação Gênica , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genoma Fúngico , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 1 , Splicing de RNA/fisiologia , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/crescimento & desenvolvimento
8.
BMC Genomics ; 7: 119, 2006 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-16712725

RESUMO

BACKGROUND: Large scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts. RESULTS: The S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences--567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were subjected to digital Northern analysis and results on expression of 5 verified by qRT-PCR. CONCLUSION: 13% of the 1965 ESTs showing identity to the A. gambiae genome sequence represent novel genes. These, together with untranslated regions (UTR) present on many of the ESTs, will inform further genome annotation. We have identified 23 genes encoding products likely to be involved in regulating the cellular oxidative environment and 25 insect immunity genes. We also identified 25 genes as being up or down regulated following blood feeding and/or feeding with P. berghei infected blood relative to their expression levels in sugar fed females.


Assuntos
Anopheles/genética , Regulação da Expressão Gênica , Insetos Vetores/genética , Abdome , Animais , Anopheles/metabolismo , Anopheles/parasitologia , Sangue , Northern Blotting , Carboidratos/administração & dosagem , Ingestão de Alimentos , Etiquetas de Sequências Expressas , Feminino , Biblioteca Gênica , Genes de Insetos , Insetos Vetores/metabolismo , Insetos Vetores/parasitologia , Plasmodium berghei , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência
9.
Genetics ; 169(4): 1915-25, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15716499

RESUMO

Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that approximately 3% of all approximately 5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders.


Assuntos
Regulação Fúngica da Expressão Gênica , Técnicas Genéticas , Genoma Fúngico , Análise de Sequência de DNA/métodos , Alelos , Proliferação de Células , Meios de Cultura/química , Meios de Cultura/metabolismo , Deleção de Genes , Genes Dominantes , Heterozigoto , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo
10.
BMC Genomics ; 6: 5, 2005 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-15651988

RESUMO

BACKGROUND: Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution. RESULTS: In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results. CONCLUSION: The expression patterns and annotation of the genes in these three groups (Early, Middle, and Late genes) are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity.


Assuntos
Anopheles/genética , Anopheles/metabolismo , Regulação da Expressão Gênica , Animais , Análise por Conglomerados , Biologia Computacional/métodos , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Feminino , Perfilação da Expressão Gênica , Biblioteca Gênica , Modelos Estatísticos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Ovário/metabolismo , Análise de Componente Principal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Vitelogênese
11.
J Antibiot (Tokyo) ; 67(1): 89-97, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24301183

RESUMO

The increasing availability of DNA sequence data offers an opportunity for identifying new assembly-line polyketide synthases (PKSs) that produce biologically active natural products. We developed an automated method to extract and consolidate all multimodular PKS sequences (including hybrid PKS/non-ribosomal peptide synthetases) in the National Center for Biotechnology Information (NCBI) database, generating a non-redundant catalog of 885 distinct assembly-line PKSs, the majority of which were orphans associated with no known polyketide product. Two in silico experiments highlight the value of this search method and resulting catalog. First, we identified an orphan that could be engineered to produce an analog of albocycline, an interesting antibiotic whose gene cluster has not yet been sequenced. Second, we identified and analyzed a hitherto overlooked family of metazoan multimodular PKSs, including one from Caenorhabditis elegans. We also developed a comparative analysis method that identified sequence relationships among known and orphan PKSs. As expected, PKS sequences clustered according to structural similarities between their polyketide products. The utility of this method was illustrated by highlighting an interesting orphan from the genus Burkholderia that has no close relatives. Our search method and catalog provide a community resource for the discovery of new families of assembly-line PKSs and their antibiotic products.


Assuntos
Antibacterianos/biossíntese , Policetídeo Sintases/genética , Engenharia de Proteínas/métodos , Análise de Sequência de DNA , Animais , Automação , Sequência de Bases , Burkholderia/enzimologia , Caenorhabditis elegans/enzimologia , Simulação por Computador , Bases de Dados de Proteínas , Lactonas/química , Família Multigênica , Policetídeo Sintases/química
13.
Genome Biol ; 11(3): R30, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20226027

RESUMO

We systematically analyzed the relationships between gene fitness profiles (co-fitness) and drug inhibition profiles (co-inhibition) from several hundred chemogenomic screens in yeast. Co-fitness predicted gene functions distinct from those derived from other assays and identified conditionally dependent protein complexes. Co-inhibitory compounds were weakly correlated by structure and therapeutic class. We developed an algorithm predicting protein targets of chemical compounds and verified its accuracy with experimental testing. Fitness data provide a novel, systems-level perspective on the cell.


Assuntos
Algoritmos , Genes Fúngicos/genética , Aptidão Genética/genética , Genoma Fúngico/genética , Genômica/métodos , Saccharomyces cerevisiae/genética , Biologia de Sistemas/métodos , Proteínas de Transporte de Cátions/metabolismo , Clozapina/farmacologia , Proteínas de Transporte de Cobre , Sistemas de Liberação de Medicamentos/métodos , Farmacorresistência Fúngica/genética , Deleção de Genes , Genes Fúngicos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Nocodazol/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
14.
PLoS One ; 4(11): e7976, 2009 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19936243

RESUMO

BACKGROUND: Anopheles funestus is a principal vector of malaria across much of tropical Africa and is considered one of the most efficient of its kind, yet studies of this species have lagged behind those of its broadly sympatric congener, An. gambiae. In aid of future genomic sequencing of An. funestus, we explored the whole body transcriptome, derived from mixed stage progeny of wild-caught females from Mali, West Africa. PRINCIPAL FINDINGS: Here we report the functional annotation and comparative genomics of 2,005 expressed sequence tags (ESTs) from An. funestus, which were assembled with a previous EST set from adult female salivary glands from the same mosquito. The assembled ESTs provided for a nonredundant catalog of 1,035 transcripts excluding mitochondrial sequences. CONCLUSIONS/SIGNIFICANCE: Comparison of the An. funestus and An. gambiae transcriptomes using computational and macroarray approaches revealed a high degree of sequence identity despite an estimated 20-80 MY divergence time between lineages. A phylogenetically broader comparative genomic analysis indicated that the most rapidly evolving proteins--those involved in immunity, hematophagy, formation of extracellular structures, and hypothetical conserved proteins--are those that probably play important roles in how mosquitoes adapt to their nutritional and external environments, and therefore could be of greatest interest in disease control.


Assuntos
Anopheles/metabolismo , Apoptose , Perfilação da Expressão Gênica , Malária/transmissão , Animais , Biologia Computacional/métodos , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Feminino , Biblioteca Gênica , Genômica , Mali , Mitocôndrias/metabolismo , Glândulas Salivares/metabolismo , Transcrição Gênica
15.
Science ; 320(5874): 362-5, 2008 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-18420932

RESUMO

Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes ( approximately 80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.


Assuntos
Genes Essenciais , Genes Fúngicos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Meios de Cultura , Farmacorresistência Fúngica Múltipla , Deleção de Genes , Genes MDR , Genômica , Heterozigoto , Homozigoto , Redes e Vias Metabólicas/efeitos dos fármacos , Família Multigênica , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia
16.
Genome Biol ; 8(1): R5, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17210077

RESUMO

BACKGROUND: The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA. RESULTS: Polytene chromosome in situ hybridization with cDNA clones was used to place 15 unmapped scaffolds (sizes totaling 5.34 Mbp) in the pericentromeric regions of the chromosomes and oriented a further 9 scaffolds. Additional analysis by in situ hybridization of bacterial artificial chromosome (BAC) clones placed 1.32 Mbp (5 scaffolds) in the physical gaps between scaffolds on euchromatic parts of the chromosomes. The Y chromosome sequence information (0.18 Mbp) remains highly incomplete and fragmented among 55 short scaffolds. Analysis of BAC end sequences showed that 22 inter-scaffold gaps were spanned by BAC clones. Unmapped scaffolds were also aligned to the chromosome assemblies in silico, identifying regions totaling 8.18 Mbp (144 scaffolds) that are probably represented in the genome project by two alternative assemblies. An additional 3.53 Mbp of alternative assembly was identified within mapped scaffolds. Scaffolds comprising 1.97 Mbp (679 small scaffolds) were identified as probably derived from contaminating bacterial DNA. In total, about 33% of previously unmapped sequences were placed on the chromosomes. CONCLUSION: This study has used new approaches to improve the physical map and assembly of the A. gambiae genome.


Assuntos
Anopheles/genética , Genoma de Inseto/genética , Mapeamento Físico do Cromossomo , Animais , Bactérias/genética , Centrômero/genética , Cromossomos/genética , Eucromatina/genética , Polimorfismo Genético , Especificidade da Espécie
17.
Science ; 298(5591): 182-5, 2002 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-12364797

RESUMO

In tropical Africa, Anopheles funestus is one of the three most important malaria vectors. We physically mapped 157 A. funestus complementary DNAs (cDNAs) to the polytene chromosomes of this species. Sequences of the cDNAs were mapped in silico to the A. gambiae genome as part of a comparative genomic study of synteny, gene order, and sequence conservation between A. funestus and A. gambiae. These species are in the same subgenus and diverged about as recently as humans and chimpanzees. Despite nearly perfect preservation of synteny, we found substantial shuffling of gene order along corresponding chromosome arms. Since the divergence of these species, at least 70 chromosomal inversions have been fixed, the highest rate of rearrangement of any eukaryote studied to date. The high incidence of paracentric inversions and limited colinearity suggests that locating genes in one anopheline species based on gene order in another may be limited to closely related taxa.


Assuntos
Anopheles/genética , Inversão Cromossômica , Ordem dos Genes , Genes de Insetos , Animais , Anopheles/classificação , Cromossomos/genética , Sequência Conservada , DNA Complementar , Evolução Molecular , Etiquetas de Sequências Expressas , Rearranjo Gênico , Ligação Genética , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Mutação , Mapeamento Físico do Cromossomo , Especificidade da Espécie , Sintenia
18.
Science ; 298(5591): 129-49, 2002 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-12364791

RESUMO

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.


Assuntos
Anopheles/genética , Genes de Insetos , Genoma , Análise de Sequência de DNA , Animais , Anopheles/classificação , Anopheles/parasitologia , Anopheles/fisiologia , Evolução Biológica , Sangue , Inversão Cromossômica , Cromossomos Artificiais Bacterianos , Biologia Computacional , Elementos de DNA Transponíveis , Digestão , Drosophila melanogaster/genética , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Etiquetas de Sequências Expressas , Comportamento Alimentar , Regulação da Expressão Gênica , Variação Genética , Haplótipos , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Insetos Vetores/genética , Insetos Vetores/parasitologia , Insetos Vetores/fisiologia , Malária Falciparum/transmissão , Dados de Sequência Molecular , Controle de Mosquitos , Mapeamento Físico do Cromossomo , Plasmodium falciparum/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único , Proteoma , Especificidade da Espécie , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
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